A major goal of this lab is to study the structure and function of Sendai virus proteins as they relate to viral pathogenicity. Sendai virus is a murine parainfluenza virus similar in structure to the influenza virus, a virus which continues to be a major cause of human infection in modern man. Knowledge gained from studying Sendai virus may be applied to influenza. Sendai virus causes a pneumotropic infection in mice. F1-R, a pantropic variant of Sendai virus causes a systemic infection. Two factors have been identified as determinants of the difference in organ tropism of the two viruses. Enhanced proteolytic cleavability of the fusion (F) protein of F1-F has been identified as one of the determinants. Another determinant is the difference in budding of the two viruses. In bronchial epithelium wild-type virus buds predominantly from the apical domain (into the lumen), whereas F1-Rbuds from both apical and basolateral domains. Some virus is, therefore, released into the basement membrane where it can easily spread into the sub epithelial cells, gain access to the bloodstream and cause a systemic infection. The infection of monolayers of polarized MDCK cells by wild-type and F1-R variant Sendai has been used to elucidate the molecular mechanism of bipolar budding by F1-R. In F1-R infected cells the microtubule network is disrupted resulting in impaired cellular polarity. It has recently been shown that when the F1-R matrix (M) protein is expressed individually, it causes a disruption in the microtubule network that results in impaired polarized transport of F, one of the envelope glycoproteins and this, presumably, results in bipolar budding of the virus. No significant changes in the microtubule network or cell polarity are seen when the wild-type M protein is expressed. Sequence analysis of viral RNA from both wild-type Sendai virus and F1-R has shown that , as compared to the wild-type gene, there are two mutations in the F1-R M gene that result in amino acid changes in the expressed protein. The first specific aim of this project is to determine if either one or both of the mutations in F1-R M is responsible for disrupting the microtubule network. Site-specific mutagenesis will be done on the wild- type M gene to create two mutant genes, each with only one of the two mutations found in the F1-R M gene. The effects of the expression of the mutant proteins on the microtubule network and cell polarity of polarized MDCK cells will be determined. A second specific aim of this project is to begin an investigation of the molecular nature of the microtubule disruption caused by F1-R M. As a first step a direct interaction between M and tubulin or microtubules will be sought by trying to coimmunoprecipitate M using anti-tubulin antibodies or to cosediment M with taxol stabilized microtubules. In addition the relative levels of total as well as free and soluble tubulin in F1-R M expressing cells, wild-type M expressing cells, nocodazole or taxol treated cells, and control cells will be determined. The ability of taxol to counteract the effects on the microtubule network and polarized transport in F1-R M expressing cells will be determined.